Propranolol blocks osteosarcoma cell cycle progression, inhibits angiogenesis and slows xenograft growth in combination with cisplatin-based chemotherapy

Osteosarcoma is still associated with limited response to standard-of-care therapy and alarmingly elevated mortality rates, especially in low- and middle-income countries. Despite multiple efforts to repurpose β-blocker propranolol in oncology, its potential application in osteosarcoma management remains largely unexplored. Considering the unsatisfied clinical needs of this aggressive disease, we evaluated the antitumoral activity of propranolol using different in vitro and in vivo osteosarcoma preclinical models, alone or in addition to chemotherapy. Propranolol significantly impaired cellular growth in β2-adrenergic receptor-expressing MG-63 and U-2OS cells, and was capable of blocking growth-stimulating effects triggered by catecholamines. siRNA-mediated ADRB2 knockdown in MG-63 cells was associated with decreased cell survival and a significant attenuation of PPN anti-osteosarcoma activity. Direct cytostatic effects of propranolol were independent of apoptosis induction and were associated with reduced mitosis, G0/G1 cell cycle arrest and a significant down-regulation of cell cycle regulator Cyclin D1. Moreover, colony formation, 3D spheroid growth, cell chemotaxis and capillary-like tube formation were drastically impaired after propranolol treatment. Interestingly, anti-migratory activity of β-blocker was associated with altered actin cytoskeleton dynamics. In vivo, propranolol treatment (10 mg/kg/day i.p.) reduced the early angiogenic response triggered by MG-63 cells in nude mice. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agent cisplatin. Sustained administration of propranolol (10 mg/kg/day i.p., five days a week), alone and especially in addition to low-dose metronomic cisplatin (2 mg/kg/day i.p., three times a week), markedly reduced xenograft progression. After histological analysis, propranolol and cisplatin combination resulted in low tumor mitotic index and increased tumor necrosis. β-blockade using propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness. Further translational studies of propranolol repurposing in osteosarcoma are warranted.


Results
First, in order to evaluate in vitro OSA sensitivity to PPN, semiconfluent monolayers of rapidly growing MG-63 and U-2OS cells were exposed to tested compound using a concentration range between 1 µM and 1 mM, based on previously reported preclinical studies [10][11][12] . After a 72 h exposure to PPN, we observed a dose-dependent cell growth inhibition in both tested cell lines obtaining IC 50 values of 45.6 and 47 µM, respectively (Fig. 1a). It is worth noting that this cytostatic activity was achieved in the absence of β-AR endogenous agonists. ADRB2 expression was confirmed in MG-63 and U-2OS cells by qRT-PCR, with MG-63 cells yielding higher gene expression values (Fig. 1b). Human prostate PC-3 cells, known to overexpress ADRB2, were used as an internal positive control 19 . In addition, human glioma U-87MG cells were used as a negative control 20 . In order to evaluate nonspecific effects of PPN, we exposed ADRB2-negative U-87MG cells to PPN and assessed its impact on cellular growth. Interestingly, no gross off-target cytostatic activity was observed in U-87MG cells after a  www.nature.com/scientificreports/ 72 h exposure to PPN at 50 µM (≈ IC 50 in responsive cells) (Fig. 1c). MG-63 cells were transiently transfected with ADRB2-targeting or non-targeting scrambled siRNAs, and treated with PPN in order to confirm ADRB2mediated antiproliferative effects. First, ADRB2 knock-down caused a significant attenuation of PPN in vitro effect. When compared to scrambled-transfected cells, ADRB2 depletion resulted in a relative 54% reduction of PPN cytostatic activity (Fig. 1d). Furthermore, growth capacity of MG-63 cells transfected with ADRB2 siRNA was severely affected, showing a cell growth reduction of 48.6% in comparison to the scramble group (Fig. 1d). ADRB2-positive MG-63 cells were then incubated with epinephrine (EPI) and norepinephrine (NOR) with the aim of evaluating their effect on cellular growth. Both catecholamines are direct-acting and non-selective α and β-adrenergic agonists. After a 72 h stimulation, EPI and NOR significantly increased OSA cell growth, showing a maximum growth-stimulatory effect at 10 nM (Fig. 1e). Interestingly, incubation with selective ADRB1 agonist dobutamine had no impact on MG-63 cell growth rate (Fig. S2). ADRB2 is a Gs-coupled receptor known to activate downstream mitogen-activated protein kinase (MAPK)-associated signaling pathways 21,22 . In order to further explore catecholamine stimulatory activity on OSA growth, we examined extracellular signal-regulated kinase (ERK) phosphorylation in MG-63 cells after ADRB2 activation by EPI and NOR (10 nM). As a result a significant increase in the phosphorylated ERK/total ERK ratio was observed after 30 and 60 min stimulation with catecholamines (Fig. 1f). As shown in Fig. 1g, addition of PPN (10 µM) to EPI plus NOR (both at a final concentration of 10 nM) was capable of completely blocking the growth-stimulating activity of catecholamines. Interestingly, growth inhibition was greater in OSA cell cultures exposed to PPN and catecholamines, in comparison to PPN alone (18% versus 54%, respectively). Similar findings were previously reported 23 , and despite the fact that the specific mechanisms remain unclear, this results could be linked to co-expression of α-2A adrenoceptor (ADRA2A) in MG-63 cells 16 , which, in contrast to ADRB2 receptor, is associated with inhibitory responses in noradrenergic neurons 24 and also with antitumor signaling in cancer cells 25 .
Next, with the aim of exploring potential mechanisms associated with cell growth inhibition by PPN we conducted cell cycle phase distribution analysis in OSA cells. A significant arrest in the G 0 /G 1 cell cycle phase and subsequent decline in both S and G 2 /M phases were observed in both MG-63 and U-2OS cells following a 24 h treatment with PPN (50 µM), as evaluated by flow cytometry studies (Fig. 1h). Cyclin D1 (CCND1) serves as a central regulator for cell cycle progression, and its overexpression results in dysregulated cyclin-dependent kinases activity, rapid cell growth under conditions of restricted mitogenic signaling, and ultimately, tumor growth 26 . As shown in (Fig. 1i), cell cycle blockade was associated with a significant reduction in CCND1 expression, as its relative gene expression values plummeted by 80% in PPN-treated MG-63 cells in comparison to vehicle-treated cells. To discard any short-term direct cytotoxicity of PPN on such treatment schedule, semiconfluent OSA cell cultures exposed to PPN were assessed by the trypan blue dye exclusion assay, showing that PPN at ≈ IC 20 and IC 50 concentrations had no effect on cell viability (data not shown). Interference of cell cycle progression after β-blockage was further confirmed by in vitro mitotic index calculation in MG-63 and U-2OS cultures exposed to DAPI for DNA and chromosome staining. Mitotic bodies that were included for quantification were representative of prophase through telophase. PPN caused a significant inhibition in the % of mitotic cells in MG-63 cultures (Fig. 1j). In U-2OS cultures, which exhibit notorious lower mitotic rates, a reduction of approximately 60% in the number of mitosis was also observed, but no statistical significance was achieved. Despite the large amount of preclinical/clinical data reporting its pro-apoptotic effects on malignant cells 9,11 , PPN at a concentration ≈ IC 50 was not capable of inducing apoptosis in MG-63 or U-2OS cells, as evaluated by quantification of hypodiploid sub-G 0 /G 1 cell populations and TUNEL labeling ( Fig. 1k and l).
Early metastatic spread and recurrences after tumor removal are strongly related to poor prognosis in OSA 27 . Considering that tumor cell migration, colony establishment and microtumor outgrowth are all hallmarks of the early stages of metastatic progression, we tested the effect of β-blocker PPN on the establishment and progression of 2D tumor colonies, 3D spheroid growth, OSA cell chemotaxis and vascular morphogenesis. Clonogenic growth of OSA cells was severely suppressed after 7 days of incubation with 1, 10 and 50 µM of PPN, reducing colony forming capacity by 31, 46 and 100% in MG-63 cells and 8, 27 and 96% in U-2OS cells, respectively, in comparison to vehicle-treated cells (Fig. 2a) (IC 50 = 19.26 and 25.28 µM for MG-63 and U-2OS cell lines, respectively). The latter IC 50 values are substantially lower than those previously observed in cell growth assays (approximately 45 µM for both tested cell lines), showing that OSA cells are particularly sensitive to prolonged PPN exposure, especially during colony establishment and outgrowth. Additionally, metabolic activity of MG-63 colonies was assessed using the MTS assay in which exposure to PPN (1, 10 and 50 µM) reduced proliferation by 25, 36 and 94%, depending on drug concentration (Fig. S3). As above-mentioned, anti-OSA activity was also evaluated on established multicellular spheroids during a 7 day treatment period. MG-63 cells were chosen for their capacity of forming homogeneous and rapidly-growing spheroids. In contrast to control spheroids which grew until reaching a sixfold increase in volume, growth of PPN-treated spheroids was completely blocked (Fig. 2b, i and ii), as observed in representative photographs. Chemotaxis of MG-63 and U-2OS cells after an overnight exposure to PPN (50 µM) was also significantly impaired, causing a cell migration reduction of 48 and 39%, respectively (Fig. 2c). Capillary-like tube formation on Matrigel-coated substrates was also evaluated in MG-63 cells as a measure of vasculogenic activity. This phenomenon, also known as vascular mimicry, is highly dependent on enhanced cell motility and migratory properties 28 , and is associated with worse prognosis in OSA 29 . As shown in Fig. 2d, PPN (50 µM) treatment was capable of effectively impairing tube formation in MG-63 cells, reducing the number of capillary-like structures by nearly 70%.
It is well established that epidermal growth factor (EGF) induces cytoskeleton reorganization and cell migration in OSA cells 30,31 . Moreover, EGF receptor (EGFR), and its related downstream pathways, are particularly overexpressed in the MG-63 cell line in contrast to other OSA cell lines 31 . Therefore, in order to evaluate whether PPN could interfere with EGF-induced actin polymerization in OSA cells characterized with high functional levels of EGFR, we treated serum-starved MG-63 cells with PPN for 1 h and then stimulated the cells for 20 min with EGF (see "Methods" section). As shown in Fig. 3 In OSA, as in many other aggressive cancers, angiogenesis and overexpression of pro-angiogenic markers correlate with disease progression 32 . In this setting, and considering the large amount of data that supports the angiostatic profile of PPN, we evaluated the effect of PPN administration on in vivo OSA-induced angiogenesis (Fig. 4). Subcutaneous plugs containing a commercial preparation of purified basement membrane matrix in addition to MG-63 cells and heparin were generated in athymic mice, and early vascular response was assessed by hemoglobin quantification after 1 week of plug implantation (see "Methods" section). Animals received PPN at a dose of 10 mg/kg/day i.p., or vehicle, for five consecutive days. As observed in Fig. 4, PPN administration was capable of impairing OSA cell-induced early angiogenic response, even after a one-week treatment, reducing by 34% the hemoglobin content in s.c. plugs, in contrast with saline vehicle-treated animals (Fig. 4a,b).
DNA-damaging agent CDDP remains as a cornerstone in the treatment of OSA 2 . With the aim of evaluating potential therapeutic benefits after addition of PPN to metronomic chemotherapy, we first conducted in vitro combination studies exploring the cytostatic effects of PPN plus CDDP (see "Methods" section). After calculating IC 50 value for CDDP as a monotherapy (Fig. 5a), sub-and supra-IC 50 concentrations of PPN and CDDP were defined for combination assays. Synergy (CI < 1) was observed in both low-and high-concentration combination scenarios after addition of PPN to CDDP (Fig. 5b,c), obtaining combination indexes of 0.36 and 0.55 for the PPN 10 µM + CDDP 1 µM, and PPN 50 µM + CDDP 10 µM combinations, respectively. Corresponding dose-reduction indexes were found as 3.96 and 1.87, respectively. Representative images of capillary-like tubular structures (identified in red lining) in different experimental conditions (×100 magnification). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ANOVA followed by Tukey's test for (a), unpaired two-sided t test for (b) and (c) and Mann-Whitney test for (d). www.nature.com/scientificreports/ Taking these results into consideration, the anti-OSA activity of PPN in combination with CDDP was further evaluated in vivo on MG-63 xenografts (Fig. 6a). Animals received i.p. PPN at a dose of 10 mg/kg in a 5-day-on/2day-off schedule, as a monotherapy or in combination with low-dose metronomic CDDP using 2 mg/kg i.p. three times per week until the end of the protocol. This scheduling differs with conventional dosing schemes which rely on few cycles of high-dose chemotherapy which are often interrupted by drug-free intervals and detoxification protocols [33][34][35] (see Methods section). Treatment with PPN in addition to sustained low-dose CDDP significantly reduced OSA xenograft progression, slowing tumor growth rate by 70% in comparison to vehicle-treated mice and significantly improving the anti-OSA activity of PPN or CDDP alone (Fig. 6b). PPN treatment as a single agent was also capable of reducing tumor growth, causing a less profound reduction of about 20%. Therapeutic benefits of PPN plus CDDP were confirmed after measuring final tumor burden, where mean tumor weight was reported as 146.7 ± 38.9 mg (mean ± SD) in the control group in comparison to 53 ± 23.8 mg in the PPN plus CDDP group (Fig. 6c). It is important to highlight that, despite observing significant effects in terms of tumor growth reduction, all tumors progressed on treatment, regardless of therapeutic intervention. All treatments were well tolerated as no significant changes in animal body weight, food or water consumption were observed throughout the protocol (Fig. 6d). Representative OSA tumor-bearing mice from different experimental groups are depicted in Fig. 6e.
Given that high mitotic counts are associated to OSA aggressiveness and poor prognosis 36 , mitotic index was assessed in resected tumors obtaining values of 8.3 ± 2.7, 5 ± 2.3, 3.36 ± 2.1 and 2.81 ± 2.2 (mean ± SD) mitotic bodies per high power field for control, PPN, CDDP and PPN + CDDP groups, respectively ( Fig. 7a,b). As shown in Fig. 6a, although all tested therapies achieved significant reductions on mitotic counts, sustained PPN + CDDP therapy yielded the most potent effect (67% inhibition in comparison to vehicle-treated group). Interestingly, despite not being reflected on its effect on OSA growth, sustained low-dose CDDP treatment was linked to an evident reduction in the proliferating status of OSA cells within the tumor, quite equivalent to combined therapy. Considering that assessment of tumor necrosis in OSA has become a relevant tool for evaluating response to therapy 37,38 , adjusted tumor necrotic rates (ATNR) were calculated for all experimental groups ( Fig. 7c) (see Methods section). Only PPN plus CDDP-based therapy was capable of significantly enhancing ATNR in comparison to vehicle-treated animals, from 58.32 ± 5.04 to 81.74 ± 2.25%, respectively. Besides a marked increase in ATNR, tumors belonging to PPN + CDDP-treated animals displayed a peculiar diffuse and rather centered pattern of viable tumor tissue in contrast to control, PPN or CDDP groups, where viable tumor tissue areas were found as large and scattered basophilic regions, interrupted by vast areas of necrotic tissue (Fig. 7d).

Discussion
To the best of our knowledge this is the first study to report the therapeutic benefits of non-selective β-adrenergic antagonist PPN in combination with chemotherapy in human OSA experimental models. Exposure of OSA cells to PPN was associated with reduced cell growth and mitosis, G 0 /G 1 cell cycle arrest and impairment of 2D/3D tumor colony formation and growth, among other effects. Similar IC 50 values of approximately 50 µM were obtained for PPN on both ADRB2-expressing MG-63 and U-2OS cell lines, confirming equivalent sensitivities for the compound. After analyzing other previously published studies, obtained IC 50 values were equivalent or, in some cases, substantially lower in comparison to other aggressive pediatric tumor types (IC 50 ranging from 114 to > 200 µM) 11,12 . However, a large number of recently reported preclinical studies focused specifically on β-adrenergic signaling consistently used a 50 µM concentration for characterizing the antineoplastic profiles and mechanisms of action of both non-selective and selective β-blockers in a wide variety of tumor types 22,[39][40][41] .
PPN direct cytostatic activity showed to be ADRB2-dependent. Silencing of ADRB2 using targeting siRNA significantly attenuated the inhibitory effects of PPN on OSA cell growth compared to scrambled-transfected control cells. Nonetheless, considering that the attenuation of the effect was not total, other off-target or non-specific alternative mechanisms cannot be completely ruled out, especially when PPN is used at high concentrations. Also  OSA cell growth seems to be influenced by catecholamines. Exposure to low concentrations of non-selective adrenergic agonists EPI and NOR resulted in increased OSA growth and activation of the ERK1/2-MAPK signaling pathway, in accordance to previously reported data 21,22 . Besides confirming that exposure of ADRB2positive OSA cells to endogenous catecholamines results in growth stimulation, here we show that MG-63 growth inhibition was greater in cell cultures exposed to PPN and catecholamines in contrast to PPN alone. The same result was observed in a previous study published by Coelho et al. in which adrenergic signaling was evaluated in colorectal cancer cells 23 . This happened with non-selective β-blockers PPN and carvedilol, but not with specific ADRB1 and ADRA1 antagonists. Despite the specific mechanism being unclear, we hypothesize that the www.nature.com/scientificreports/ enhanced growth-inhibiting effect of PPN plus catecholamines, in comparison to PPN alone, could be related to the fact that MG-63 cells co-express ADRA2A in addition to ADRB2 16 . ADRA2A acts as a presynaptic autoinhibitory receptor in noradrenergic neurons 24 , but is also expressed in some tumor types 25,45 . Different authors have demonstrated that high expression of ADRA2A was associated with a better prognosis, and its overexpression was capable of suppressing tumor invasion and growth through inhibition of the PI3K/Akt/mTOR pathway 25,45 . Therefore, as both ADRA2A and ADRB2 receptors are co-expressed in MG-63 cells, and ADRA2A is associated to antiproliferative signaling upon stimulation in malignant cells, ADRB2 blockade by PPN and ADRA2A activation by present catecholamines could collaborate into a greater tumor cell growth inhibition of MG-63 cells.
Interestingly, no significant proapoptotic activity of PPN on OSA cells was observed at 50 µM. However, it is worth noting that apoptosis induction in other preclinical studies using PPN on different tumor models was achieved using concentrations in the range of 100-300 µM 9,11,12 . Exploring such concentrations goes beyond the scope of the present work given that, according to early pharmacokinetics reports, achievable plasma concentrations of PPN are in the low µM range 46,47 .
Growth inhibition and impairment of colony formation in both MG-63 and U-2OS cells were associated with cell cycle blockade in the G 0 /G 1 phase, and a significant suppression of cell cycle regulator cyclin D1 (CCND1). These results are in accordance with previous data reported by Montoya et al., who identified cyclin D1 (among a large panel of cyclin proteins) as a key mediator of PPN inhibitory effect on mitogenic potential of breast cancer cells 9 . PPN cytostatic activity in ADRB-expressing malignant cells was also previously associated to the down-regulation of the AKT/mammalian target of rapamycin (mTOR) and the MEK/ERK1/2 MAPK signaling pathways [9][10][11] . Interestingly, with the aim of shedding some light into the largely unknown molecular landscape of OSA, genomic and transcriptomic studies have recently identified frequently mutated genes and copy number alterations in OSA samples 48,49 . Besides confirming TP53, RB1 and MYC as driver genes, it was reported that PI3K/AKT/mTOR and MAPK pathways are heavily altered in OSA and are central players in disease initiation and progression. PPN reduced OSA cell migratory capacity and tubule formation, and impairment of chemotaxis was associated with altered cytoskeleton dynamics and reduction of EGF-triggered actin polymerization. In this context, it is recognized that EGF-induced cell migration and EGFR activation in OSA cells are also associated to phosphorylation of ERK1/2, AKT, S6, and GSK3β, confirming activation of MAPK and PI3K/Akt downstream signaling pathways 30,31 . According to these data, PPN could be capable of blocking key protumoral signaling pathways involved in OSA growth and motility. However, the specific molecular mechanisms underlying these effects remain to be determined and further validated.
In our study, PPN anti-OSA activity was related to direct cytostatic effects and cell cycle arrest, decreasing survival and mitosis in ADRB2 expressing tumor tissue. However, SNS activation and β-adrenergic signaling can regulate a wide range of cancer-associated molecular pathways via both direct effects on ADRB expressing transformed cells and regulation of other ADRB-bearing cells populating the complex tumor stroma, such as adipocytes, fibroblasts, and vascular and immune cells 50,51 . In spite of using T cell-deficient nude mice for our in vivo studies, this animal model still has a robust innate immune response involving dendritic cells, tumor-associated macrophages and natural killer cells, which are known to play a key role against OSA 52 . Taking these concepts www.nature.com/scientificreports/ into account, other unexplored microenvironmental mechanisms associated with pleiotropic β-blockade, such as increased immune function, reversal of adipose tissue-driven inflammation or cancer-associated fibroblast inactivation, could be playing a part in the overall PPN activity and should not be discarded 53,54 . It is important to note that in the current study, concentrations in the range of 10-50 µM PPN were needed in order to significantly impair OSA aggressiveness in vitro. As mentioned, these concentrations are not achievable in vivo with standard dosage (plasma concentrations in PPN-treated patients can reach values of up to 500 ng/mL, or ≅ 2 µM) 46,55 . This suggests that the in vivo anti-tumor efficacy of PPN may not be the result of isolated direct cytostatic activity against malignant tissue, but rather through the sum of additional stroma-related mechanisms, such as angiogenesis inhibition. In accordance with an overwhelming body of evidence describing PPN as a highly efficient angiostatic agent 8,56 , we demonstrated that PPN, administered following a clinically-relevant dosage scheme of 10 mg/kg/day i.p., reduced early angiogenic response in MG-63 implants. Enhancement of antineoplastic activity as a result of combining PPN with cytotoxic agents was previously reported for other tumor types, in which addition of repurposed drug to chemotherapy increased antiangiogenic and antiproliferative effects of monotherapies, as well as decreased distant metastases in patients [57][58][59] . Moreover, it was recently reported that β-blockade increases sensitivity to radiotherapy in different bone sarcoma models, especially in canine OSA 17 . In our in vivo protocol, sustained treatment with PPN using validated human equivalent doses (10 mg/kg/day i.p.), in addition to low-dose metronomic CDDP (2 mg/kg/day i.p.), significantly reduced the progression rates of OSA tumors, enhancing therapeutic benefits without increasing toxicity. Enhanced antineoplastic effectiveness of PPN and CDDP combination therapy could be associated with multiple mechanisms. First, it is known that low doses of CDDP induce cell cycle arrest in OSA, especially in highly proliferating MG-63 cells 60,61 , and that sensitivity of MG-63 tumors to CDDP therapy is highly dependent on cyclin D1 signaling 62 . Secondly, low-dose metronomic scheduling of CDDP is known to block tumor-driven angiogenesis triggering angiostatic mechanisms via modulation of TIMP-1 and different MMPs [63][64][65] . Considering the effects of PPN on angiogenesis and cell cycle progression, combination of this β-blocker with CDDP could favor a www.nature.com/scientificreports/ cooperative suppression of mitogenic potential of OSA cells as well as an inhibition of tumor-associated vasculature, resulting in impaired xenograft growth, reduced mitosis and enhanced tumor necrosis. Other potential mechanisms, such as modulation of P-gp function, could be playing a role in the observed cooperative benefits and should be considered in the future 40 . These findings bear high translational relevance given that necrosis rate in response to therapy and mitotic index determination are two histopathological parameters routinely used in the clinical management of OSA, linked to disease aggressiveness and prognosis 36,66 . Our study has several limitations worth noting. It is widely recognized that in vivo models should recapitulate the tumor stroma as closer to the clinical setting as possible 67 . Given that PPN targets both tumor and its microenvironment, the influence of OSA tumor stroma should not be neglected, and the use of orthotopic paratibial or intraosseous tumor models should be considered for future studies. In addition, it is widely accepted that OSA is characterized by a complex genotype and high inter-and intra-tumor heterogeneity 68 . As a consequence, with the aim of reproducing this complex clinical heterogeneity, the use of OSA patient-derived xenografts should be addressed as a priority. Finally, although we show that PPN treatment affects different metastases-associated cellular traits in vitro, such as 2D/3D colony growth or tumor cell chemotaxis, effects of PPN administration on in vivo metastatic spread and outgrowth, especially to lungs, should be evaluated in order to fully characterize its therapeutic potential in OSA.
OSA is fatal for about one-third of the patients worldwide. However, prognosis is significantly poorer for LMICs 69 . Higher mortality rates are mainly due to economic inequalities and inefficient health-care systems, resulting in diagnosis at advanced stages and poor access to treatment resources, including surgery, radiotherapy and high-cost novel cancer drugs 70 . As previously introduced, metronomics combines drug repurposing with metronomic chemotherapy and could act as a substitute for standard treatments when unavailable or undoable 69 . PPN is a low-cost widely-available FDA-approved drug and its repositioned use in oncology is gaining strength as high quality preclinical and clinical evidence accumulates. On the other hand, in the past years metronomic chemotherapy has emerged as a promising multi-targeted therapeutic approach, showing clinical benefits in aggressive pediatric tumors with low associated-toxicities and used in outpatient settings [71][72][73][74][75] . Therefore, the www.nature.com/scientificreports/ combination of metronomic chemotherapy with repurposed PPN seems to deserve further research in OSA especially in developing countries. This approach could not only improve the effectiveness of chemotherapy but also contribute to reducing severe CDDP-related adverse effects.

Conclusions
Survival rates of OSA patients have remained practically unaltered in the last 30 years, highlighting the need of intensifying research efforts and drug development programs to tackle this disease. β-adrenergic antagonization seems an achievable and interesting therapeutic approach to inhibit OSA progression (Fig. 8). Integration of novel therapeutic approaches such as the use of repurposed PPN into current OSA management is both promising and challenging. We believe that this study could help lay the groundwork for translating the use of PPN as a novel adjuvant therapy in OSA.

Methods
Drugs and compounds. PPN hydrochloride lyophilized powder (Sigma-Aldrich, Missouri, USA) was first solubilized using citrate buffer (pH 3) and further diluted with phosphate buffer saline (PBS) to reach working concentrations. CDDP was purchased from Abbot laboratories (Illinois, USA). Catecholamines EPI and NOR were purchased from BIOL laboratories (Buenos Aires, Argentina).  www.nature.com/scientificreports/ OSA cell growth. Crystal violet assay was used for assessing OSA cellular growth. Briefly, MG-63, U-2OS or U-87MG cells were plated in 96-well flat bottom plates at a density of 2.5 × 10 3 or 5 × 10 3 , respectively, per 100 µL in complete DMEM, allowed to attach overnight, and then treated with PPN, catecholamines, dobutamine, CDDP or vehicle for 72 h. Then cells were fixed for 10 min with methanol and then stained with 0.5% crystal violet solution for another 10 min. Following a washing step with deionized water, fixed cultures were dried overnight. Quantification was performed by direct counting or absorbance measurement (λ = 595 nm) using a multiplate reader after adding an ethanol/acetic acid 3:1 solution. Values are presented as a percentage of control. For the calculation of the half maximal inhibitory concentration (IC 50 ), linear and nonlinear regressions were applied, depending on optimal curve-fitting. For synergy assessment in drug combination studies, the nature of the interactions between studied compounds was analyzed using the combination index (CI) method calculated in the Compusyn software v1.0 (Combosyn Inc., New Jersey, USA, www. combo syn. com). For the quantitative evaluation of this interaction we used full-range dose-response curves for each evaluated drug. Synergy was determined when CI was < 1.

Tumor cell lines. Human
Transient transfection with ADRB2-targeting siRNA. For transfection, 2.5 × 10 3 MG-63 cells were seeded per well in 96 well-plates in complete medium. After 24 h, knock-down was conducted using double stranded small interfering RNA (siRNA) targeting ADRB2 as previously described 76,77 . For increased knockdown efficiency, a 10 nM mixture of three different sequences of ADRB2-targeting siRNA was used. Non-targeting scrambled siRNA at 10 nM was used as a control. ADRB2 siRNAs and non-targeting controls were kindly Western Blot. MG-63 cells were plated on 6-well culture dishes in growth medium. After 24 h starvation, cells were treated for 5, 10, 30, 60 or 120 min with 10 nM of catecholamines EPI and NOR. After treatment, cells were washed twice with PBS and protein extracts were prepared by homogenizing an equal number of cells in ice-cold RIPA buffer containing protease and phosphatase inhibitors (P8340 and P0044, Sigma-Aldrich, Missouri, USA). Protein concentrations were determined by Bradford assay. Samples were separated by 10% SDS-PAGE and electro-transferred onto polyvinylidene difluoride membranes. After blockade with 5% non-fat milk in TBST buffer, membranes were incubated with primary antibodies at 4 °C overnight, followed by anti-rabbit IgG 1:1000 horseradish peroxidase (HRP)-conjugated secondary antibody (7074, Cell Signaling Technology Inc., Massachusetts, USA) for 1 h. Immunoreactive signals of target proteins were detected with ECL™ Prime Western Blotting System (Cytiva RPN2232, Sigma-Aldrich) and images were taken using the ChemiDocChemiluminescence Western blot Scanner (Bio-Rad Laboratories Inc., California, USA) and analyzed with Image Lab software (Bio-Rad Laboratories Inc. Animals. Outbred athymic female N:NIH(S)-nu mice aged 8 weeks with a weight of approximately 23 g, were purchased from the School of Veterinary Sciences Animal Facility at National University of La Plata (Buenos Aires, Argentina) and, after randomization, housed at 5 mice per cage in our animal facility at the National University of Quilmes. Food and water were provided ad libitum and the general health status of the animals was monitored daily.
Modified Matrigel plug assay. To evaluate the effects on OSA-induced angiogenesis, a modified Matrigel plug assay was conducted. A mixture containing 400 µl of Matrigel, heparin (50 U/ml) and 4 × 10 6 MG-63 cells in 100 µl serum-free DMEM medium was injected subcutaneously into athymic mice. Treatment consisted of five daily consecutive doses of PPN 10 mg/kg i.p. This clinically relevant dosing scheme was adopted from previously reported preclinical studies 59 and is in range with clinical references (including pediatric population) 84 after "human to mice" dose conversion. This calculation is performed by dose extrapolation using the 'dose by factor' method based on allometric scaling, following the United States Food and Drug Administration guidelines 85 . Animals were sacrificed 7 days after cell injection. Plugs were recovered and the extent of vascularization was assessed by the amount of hemoglobin detected in the implants using the Drabkin method (Sigma-Aldrich, St Louis, MO, USA) 86,87 . The mean optical density of plugs from the control group was taken as 100% (relative hemoglobin content). Representative plugs were photographed before processing for hemoglobin quantification.
OSA xenograft progression. Human OSA tumors were heterotopically generated after subcutaneous injection of a 150 µl suspension containing 5 × 10 6 MG-63 cells in DMEM and Matrigel (Corning, New Jersey, USA) in a 2:1 volume ratio MG-63 cells in athymic mice 88 . Tumors were measured periodically with a caliper and tumor volume was calculated by the formula: 0.52 × width 2 × length. During the protocol animal weights and tumor growth rates (TGR) were also assessed. TGR represents the slopes of the linear regressions of the tumor volumes over time. Treatment schedules started 3 days after cell inoculation, when tumors were detected by palpation. When first signs of skin infiltration appeared on primary lesions and larger tumors reached a volume of 300 mm 3 , animals were photographed and protocol was ended. Animals were euthanized by cervical dislocation and tumors were removed, weighted, fixed with formalin and processed for hematoxylin and eosin (H&E) staining.
In vivo combination studies were performed by administering PPN (10 mg/kg i.p.) in a 5-day-on/2-day-off schedule, alone or in combination with CDDP (2 mg/kg i.p.) during 4 weeks. Once again, PPN 59 and CDDP 89 dosage was defined according to previously reported preclinical studies. Chemotherapy was used following a metronomic scheduling consisting in sustained administration of low-dose CDDP three times per week during 4 weeks, without drug-free intervals until the end of the protocol 35,64 . Tumor growth rates and volume, as well as total animal weight were recorded or calculated throughout the protocol. Histopathological assessment of OSA tumors involved mitotic index quantification in viable sections of H&E-stained tumor slides and determination of adjusted tumor necrotic rate after treatment 32 . Mitotic bodies in H&E-stained slides were counted in 16 randomly-selected HPF (×400). Only viable sections of tumor tissue were analyzed for mitotic index calculation. Histological analysis was performed and confirmed by two blinded researchers. For tumor necrosis assessment, color brightfield images of entire H&E-stained tumor sections were acquired at ×2.5 magnification using a Cytation Gen5 Reader (BioTek, Vermont, USA). Images were collected using a 4 × 5 grid and the stitching was performed with the "Image Montage" function setting a tile overlapping of 10%. Necrotic area in tumor tissue sections was measured using ImageJ 1.5j8 Software (NIH, Maryland, USA, imagej.nih.gov). Tumor necrosis was identified as tissue areas with a marked increase of eosinophilia and quantification of both necrotic area (NA) and viable area (VA) was performed using the "Color Threshold'' tool. Tumor necrotic rate (TNR) was then calculated as: "TNR = (NA * 100) / (VA + NA)" in 4 sections per experimental group. Adjustment of % of necrotic areas to changes in tumor size and determination of adjusted tumor necrotic rate (ATNR) was performed using the following equation: "ATNR = 100-(100-TNR) x RTGR", where RTGR stands for group-specific relative tumor growth rates. RTGR was obtained after transforming TGR values, taking the TGR of the control group (6.8 mm3/day) as "1". As a result, RTGR of 0.75, 0.94, and 0.34 were respectively used for necrosis adjustments in PPN, CDDP and PPN + CDDP-treated tumor slides.
Statistics. Statistical analysis was performed using the GraphPad Prism v6.0.0 (GraphPad Software Inc., San Diego, CA, USA, www. graph pad. com) or Compusyn software v1.0 (Combosyn Inc., New Jersey, USA, www. combo syn. com). To compare differences between two experimental groups Mann Whitney or t tests were used for non-parametric or normal distribution of data, respectively. In case of more than two experimental groups, ANOVA analysis with Tukey's multiple comparisons post-test was used when normal distribution of data was determined. Kruskal-Wallis analysis with Dunn's multiple comparisons post-test was used in case of non-parametric distribution of data. Differences were considered statistically significant at a level of p < 0.05. Data corre- www.nature.com/scientificreports/ sponds to at least 2 or 3 independent experiments unless stated otherwise. Data were presented as mean ± standard deviation (SD) or standard error of mean (SEM).
Ethical approval. All

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.